Approximately 2,000,000 people are infected with hepatitis C virus (HCV) in Japan. It is highly probable that HCV infection causes chronic hepatitis C. Chronic hepatitis C leads to liver cirrhosis and hepatoma, which are lethal diseases, in from 10 to 20 years.
There are several tens of genotypes of HCV. In Japan, ratios of genotype 1b, genotype 2a, and genotype 2b are about 70%, about 20%, and 10%, respectively. At present, therapy with pegylated-interferon (PEG-IFN) in combination with ribavirin (RBV) is standard therapy for chronic hepatitis C (covered by insurance since December 2004). Its curing ratio is 80% or more for genotype 2a or 2b, but is as low as about 55% for genotype 1b. Therapy with telaprevir (HCV protease inhibitor), in addition to the therapy with PEG-IFN in combination with RBV, is scheduled to start, and the triple therapy is expected to increase the curing ratio for genotype 1b as well to about 70 to 80%. However, the following problems have not yet been overcome: a case of discontinued therapy due to exacerbation of a side effect (anemia) of RBV by telaprevir; a case of onset of depression resulting from therapy with IFN; an ineffective case where IFN is not effective; and presence of a large number of older people that cannot be treated with IFN. Accordingly, there is a need for development of a novel anti-HCV agent that can cure the above-mentioned cases as well.
For searching an anti-HCV agent, there is mainly used a reporter assay system (Ikeda M et al., BBRC, 329:1350-1359, 2005) developed based on HCV-replicon-replicating cells (HCV subgenomic RNA autonomously replicates and proliferates in the cells), which were developed by a German group in 1999 (Lohmann V et al. Science, 285:110-113, 1999), or cells in which full-length HCV RNA with a structural region of HCV autonomously replicates (full-length HCV-RNA-replicating cells) (Ikeda M et al., J. Virol. 76:2997-3006, 2002). The inventors of the present invention developed an assay system using OR6 cells as another assay system (JP 4009732 B2). In the OR6 cells, HCV RNA is linked to RNA encoding renilla luciferase gene. Therefore, the assay system allows a level of HCV-RNA replication to be quantitatively monitored by simply measuring renilla luciferase activity, and has been markedly improved in terms of time and cost as compared to conventional RNA quantification. In the assay system using OR6 cells, an HCV-RNA replication inhibitor can be searched from a compound library or the like. Therefore, a large number of compounds have already been screened, and a plurality of compounds each exhibiting anti-HCV activity have been selected, some of which have been tested towards clinical application. The inventors of the present invention found a statin agent (Patent Literature 1: JP 2007-63284 A), teprenone and 5-HETE (Patent Literature 2: JP 2010-59080A), oncostatin M (Patent Literature 3: JP2010-59081A), and the like as anti-HCV agents by screening existing drugs and the like through use of the assay system using OR6 cells.
The above-mentioned reporter assay system is an assay system that is useful in that the level of HCV-RNA replication can be simply and quantitatively monitored. Hitherto, however, there has been a problem in that such assay system can be utilized only for cells derived from a human hepatoma-derived cell line, HuH-7 (Nakabayashi H et al., Cancer Res. 42:3858-3863, 1982). A clinical trial of a compound obtained by screening using only the HuH-7-derived assay system as an anti-HCV agent candidate involves a risk in terms of a therapeutic effect. Further, screening using cells of only one kind may miss a drug exhibiting anti-HCV activity. In order to reduce such risk, the inventors of the present invention worked on development of cells that were derived from a human culture cell line different from HuH-7 and were able to be used for an assay system, and in 2008, succeeded in developing cells (ORL8 and ORL11) derived from a human hepatoma cell line, Li23, in which the level of HCV-RNA replication can be monitored by measuring renilla luciferase activity (WO 2010/026965 A1, Kato N et al., Virus Res. 146:41-50, 2009). After that, anti-HCV agent candidates reported previously were evaluated by assay systems using OR6 cells and ORL8 cells. As a result, in about half of the cases, a 50% effective concentration (EC50) value was high 3-fold or more, or was as low as one-third or less as compared to values reported previously. In addition, also in comparison between the assay systems using OR6 cells and ORL8 cells, a drug (methotrexate) having different EC50 values up to 2,000-fold was found (Non Patent Literature 1: Ueda Y et al., BBRC, 409:663-668, 2011).
According to a previous report on artemisinin, a drug used as an antimalarial agent, an assay system using HuH-7-derived HCV-replicon-replicating cells demonstrates that artemisinin has anti-HCV activity, though the activity is weak (Non Patent Literature 2: Paeshuyse J et al., BBRC, 348:139-144, 2006). The inventors of the present invention performed assays using full-length HCV-RNA-replicating cells (e.g., OR6 cells and ORL8 cells) derived from the above-mentioned two kinds of cell lines (HuH-7 and Li23). However, artemisinin had an EC50 in the OR6 cells of 81 μM and an EC50 in the ORL8 cells of 23 μM, which were high concentrations, revealing that artemisinin was not a potential anti-HCV agent candidate (Non Patent Literature 1: Ueda Y et al., BBRC, 409:663-668, 2011). As another antimalarial agent, the inventors of the present invention reported a compound N-89 obtained by screening using antimalarial activity as an indicator (Patent Literature 4: JP 2000-229965 A, Non Patent Literature 3: Kim H-S et al., J. Med. Chem., 44:2357-2361, 2001). In addition, there is also a report that a compound N-251 has antimalarial activity (Patent Literature 5: JP 4289911 B2 and Non Patent Literature 4: Sato A et al., Parasitology Int., 60:270-273, 2011). However, those compounds have structures quite different from that of artemisinin.
There is a demand for development of a highly safe anti-HCV agent that exhibits potent anti-HCV activity without being influenced by genetic diversity of the virus.